EGFR has been demonstrated to be overexpressed in many types of human solid tumors, including lung cancer, colon cancer, breast cancer, gastric cancer, brain cancer, bladder cancer, head and neck carcinoma, ovarian cancer, esophagus cancer, liver cancer, kidney cancer and prostate cancer. The development of antibody drug for the epidermal growth factor receptor family provides an opportunity for the treatment of these tumors.
At least two antibody drugs against EGFR have been used in clinical tumor treatment, for example Erbitux® (also known as Cetuximab) and panitumumab. But the applications of these antibodies have some limitations. This is because on the one hand, EGFR is expressed in many human solid organs such as skin and liver, which may leads to the uptake of the antibody drugs by these organs after they were administered in vivo (Baselga J et al. Phase I studies of anti-epidermal growth factor receptorchimeric antibody C225 alone and in combination with cisplatin. J. Clin. Oncol. 2000 February; 18(4): 904-14, and Faillot T et al. A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas. Neurosurgery. 1996 September; 39(3): 478-83). On the other hand, nonspecific effects of these antibodies on the tissue with normal EGFR expression, may result in the side effects such as skin rash during the administration of antibody drug such as Erbitux (Agero A L, et al, Dermatologic side effects associated with the epidermal growth factor receptor inhibitors. J Am Acad Dermatol. 2006 October; 55(4): 657-70), and some serious side effects can lead to the patient to have to stop taking the drug.
In order to reduce side effects caused by the interaction between the existing EGFR antibodies and normal tissues, several monoclonal antibodies against tumor specific EGFR epitopes were developed, for example, an antibody targeting the junction LEEKKGNY generated by the deletion of 267 amino acids in exons 2-7 of de2-7EGFR (also known as EGFRvIII) (see antibody 131 disclosed in patent application PCT/US2004/020295); antibodies for cryptic epitopes of EGFR such as mAb806 and CH12 (see US patent applications US2011/0076232A1 and WO/2011/035465). When EGFR is activated, overexpressed, or mutated, its cryptic epitope (287CGADSYEMEEDGVRKC302) may be exposed and bind to antibodies such as mAb806 for this epitope (Garrett T P et al., Antibodies specifically targeting a locally misfolded region of tumor associated EGFR. Proc Natl Acad Sci USA. 2009; 106(13): 5082-7). In animal experiments, these antibodies display antitumor effects and show better tumor specificity than other anti-EGFR antibodies developed previously. Human-murine chimeric antibody ch806 which was derived from mAb806 exhibits a strong tumor targeting ability and no obvious skin toxicity was observed in phase I clinical trials (Scott A M, Lee F T et al, A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors. Proc Natl Acad Sci USA. 2007 Mar. 6; 104(10): 4071-6). Even at a dose of 5 mg/m2, ch806 displays tumor uptake. For other previous anti-EGFR antibodies, they need about 10 to 20 times of the dose to show tumor uptake (Divgi C R et al. Phase I and imaging trial of indium 111-labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinoma. J Natl Cancer Inst. 1991 Jan. 16; 83(2): 97-104). (Rushika M. Perera, et al. Treatment of Human Tumor Xenografts with Monoclonal Antibody 806 in Combination with a Prototypical Epidermal Growth Factor Receptor Specific Antibody Generates Enhanced Antitumor Activity. Clin Cancer Res 2005; 11(17): 6390-9).
Additionally, the antibodies for above-mentioned epitopes such as CH12 do not show obvious antitumor efficacy on the tumors expressing other forms of EGFR (for instance, T790M mutated EGFR). The T790M mutation often occurs a period of time after the therapy of an EGFR-related lung adenocarcinoma with small molecular tyrosine kinase inhibitors (Xu Y et. al, Acquired resistance of lung adenocarcinoma to EGFR-tyrosine kinase inhibitors gefitinib and erlotinib. Cancer Biol Ther. 2010 April; 9(8): 572-82. Epub 2010 Apr. 26).
Thus, it is valuable to reform these antibodies to increase their antitumor activities (i.e., reduce the minimum effect dose), and expand their antitumor ranges.
One of the interesting ways to increase the antitumor activities of antibody is to construct bifunctional antibody. Bifunctional antibody that specifically recognizes both EGFR and CD3 antigen has been described in the prior art. One part of its functional domain is specific to EGFR and the other part of its functional domain is specific to the CD3 antigen on T cells. Although the bifunctional antibodies made of Cetuximab or Pantitumumab and anti-CD3 antibody display excellent antitumor activities, they show relatively strong toxic effects on the normal cells or tissues with EGFR expression in primate animal experiments (Lutterbuese R, Raum T et. al, T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer cells. Proc Natl Acad Sci U.S.A. 2010; 107(28): 12605-10).
Due to the nature of complexity of biological experiments, it is not sure whether each functional domain of the prepared bifunctional antibody can retain the original antigen binding specificity and further display the antitumor activity, although technology for the preparation of bifunctional antibody already exists.
This field further requires bifunctional antibody with increased tumor killing biological activity and increased tumor recognition specificity for the EGFR-related tumors. The invention realizes this purpose.